Gibberellins (GAs) are a group of tetracyclic diterpene carboxylic acids involved in a variety of developmental processes. They were originally identified through their effect on stem elongation (Phillips, A. L., Plant Physiol. Biochem 36: 115-124, 1998), and are now implicated in all stages of the plant life cycle including seed germination, leaf expansion, floral induction, fruit maturation, and apical dominance (Harberd, N. P. et al., BioEssays 20: 1001-1008, 1998). There are at least 126 different GAs identified in plants, fungi, and bacteria; however, most are precursors or degradation products, which are inactive forms. The bioactive GAs in higher plants include GA1, GA3, GA4, and GA7 (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000).
The GA biosynthetic pathway has three different classes of enzymes that catalyze specific reactions in the synthesis of bioactive GAs: terpene cyclases, Cyt P450 monooxygenases, and 2-oxoglutarate-dependent dioxygenases (Yamaguchi, S. and Y. Kamiya, Plant Cell Physiol. 41: 251-257, 2000). The first set of reactions of the biosynthetic pathway, from trans-geranylgeranyl diphosphate (GGPP) to GA12-aldehyde, is the same in all systems that have been studied. GGPP is converted to ent-kaurene via the terpene cyclases. ent-kaurene is then oxidized by Cyt P450 monooxygenases to GA12-aldehyde, GA12 and then GA53. GA12 and GA53 are the initial substrates for the 2-oxoglutarate-dependent dioxygenases. The specific enzymatic steps for the synthesis of bioactive GAs from GA 12 are species specific.
The last reactions producing bioactive GAs and the first breakdown reactions involve several types of dioxygenases. The nomenclature of these dioxygenases is variable throughout the literature. Herein, the most commonly used name is listed first, followed by any other names also used. GA 20-oxidases remove the C-20, whereas 3β-hydroxylases (also called 3-oxidases) introduce the 3β-hydroxyl group; both are steps on the way to bioactive GAs. GA 2-oxidases (also called 2β-hydroxylases) introduce a 2β-hydroxyl group resulting in inactive products that cannot be converted to active forms (Thomas, S. G. et al., Proc. Natl. Acad. Sci. USA 96: 4698-4703, 1999). GA 2-oxidases generally act on GAs with 19 carbons, although there is evidence of 2β-hydroxylation of C20-GAs (Hedden, P. and Y. Kamiya, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 431-60, 1997).
GA-modifying enzymes produce a vast number of GAs, although most are precursors or inactive forms. Many dioxygenases have been shown to be multifunctional, catalyzing consecutive reactions in the pathway, or modifying different, but structurally similar, GAs. For example, GA5, a GA 20-oxidase, converts GA12 to GA15 to GA24 to GA9, and GA53 to GA20 (Yamaguchi, S. and Y. Kamiya, Plant Cell Physiol. 41: 251-257, 2000). This multifunctional property allows many different GAs to be formed from relatively few enzymes.
Several of the dioxygenases can be grouped into small gene families. In Arabidopsis, GA 20-oxidases and GA 3β-hydroxylases are each encoded by at least four genes, and GA 2-oxidases are claimed in one review to be encoded by at least six genes (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000). Although the three groups of dioxygenases act on similar GA substrates, cluster analysis shows that they are no more closely related to each other than to any other plant dioxygenase (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000). The identity between different groups of GA dioxygenases is approximately 20-30% within one species, such as Arabidopsis (Table 1). Within a group, however, the identity is higher, even among species. Arabidopsis GA 20-oxidases are approximately 55-70% identical to each other, and 50-60% identical to 20-oxidases of other species (Prescott, A. G. and P. John, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 245-71, 1996). The three published Arabidopsis GA 2-oxidases are 49-68% identical to each other (Thomas, S. G. et al., Proc. Natl. Acad. Sci. USA 96: 4698-4703, 1999), and 35-65% identical to GA 2-oxidases of other species (Table 2). The various members of each dioxygenase family are differentially expressed within the plant, and may be involved in different developmental processes (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000).
Chemical modification of GA levels is common in agriculture and horticulture. Seedless grapes are often treated with GA3 to increase berry size. Conversely, many crops and ornamental plants are treated with chemicals that act to inhibit enzymes in the GA-biosynthetic pathway (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000). Height reduction in ornamentals is currently achieved in many plants, such as poinsettias and petunias, via treatment with GA-inhibiting chemicals to produce compact plants that are more desired by consumers. Height reduction in a number of crop plants has resulted in increased yields and yield stability. In fact, compact crop plants have been a cornerstone of the great enhancements in agriculture yields over the past three decades. Compact plants can be grown more densely and are more resistant to storm damage (lodging) than taller wild type versions. Compact plants are easier to harvest because they hold the seed products closer together, reducing loss during harvesting.
Many groups have manipulated GA levels by transgenetically altering the expression of genes involved in GA metabolism. Overexpression of GA 20-oxidases in Arabidopsis has yielded plants with elevated GA levels which results in plants that are taller and have lighter green leaves than wild-type plants (Huang, S. et al., Plant Physiol. 118: 773-781, 1998). Suppression of GA 20-oxidases by antisense RNA has produced Arabidopsis plants that display phenotypes similar to weak GA-deficient plants; these plants have darker green cotyledons, were about 40% shorter than wild-type plants at maturity, and flowered slightly later than wild type in short-day conditions (Coles, J. P. et al., Plant J. 17: 547-556, 1999). Overexpression of a unique pumpkin 20-oxidase, which produces an inactive GA, has produced plants with a weak GA-deficient phenotype in Solanum dulcamara. These plants are semi-dwarfs, have smaller, darker green leaves, flower earlier, and produce more fruit and seed per fruit than wild type plants (Curtis, I. S. et al., Plant J. 23: 329-338, 2000). Overexpression of a bean 2-oxidase in Arabidopsis has produced plants with a variety of phenotypes including GA-like dwarfs and semi-dwarfs (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000). The same range of phenotypes was seen when the bean 2-oxidase was overexpressed in wheat (Hedden, P. and A. L. Phillips, Trends Plant Sci. 5: 523-530, 2000). Ectopic expression of a rice 2-oxidase resulted in rice plants which were dwarfed and had darker green, shorter and wider leaf blades, a typical GA-deficient phenotype for rice (Sakamoto, T. et al., Plant Physiol. 125: 1508-1516, 2001).
Genetically altering GA-modifying enzymes has the advantage of providing a means of decreasing chemical usage in plant production, as well as decreasing energy and time expenditures in chemical applications.